Project Summary/Abstract Alzheimer?s disease (AD) is the most common cause of dementia in today?s aging population, and the disease begins insidiously and silently 5-10 years before major symptoms appear (i.e., cognition impairment). However, the pathogenic mechanisms are still not fully understood, and there is no effective therapy. Neuropathological hallmarks of AD are neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau and senile plaques comprising amyloid-? peptides (A?) derived from amyloid precursor protein (APP). Interestingly, pathogenic APP processing and tau hyperphosphorylation is highly regulated by various protein kinases or phosphatases in AD. Moreover, there is a growing body of the evidence that A? deposits are associated with tau tangles. Therefore, a targeting agent that can effectively and safely ameliorate NFTs and amyloid plaques represent promising therapeutic and potential prophylactic treatments in neurodegenerative conditions including AD. In our parent R01 project NIBIB #R01EB022230, we have been developing multifunctional nanoprobes which enable targeting, imaging, and image-guided intervention of pancreatic neuroendocrine tumors (PNETs). Briefly, by engineering chemical moieties composing nanoprobes systematically, we were able deliver nanoprobes to the pancreas and achieve selective targeting of PNETs. Moreover, over 80% of the injected dose was ultimately eliminated into urine within 24 h. This narrows the chemical window to be modulated among targeting anchor, imaging moiety, and distribution domain; however, our laboratory has worked diligently to create a reciprocal arrangement whereby each chemical composition provides balancing properties to the others. Interestingly, during the evaluation of the cellular mechanism and biodistribution of the targeted nanoprobes, we found some pancreas-targeted contrast agents show great affinities in the brain tissue, specifically to the A? peptides, which is structurally similar to the death-associated protein kinase 1 (DAPK1) inhibitor. DAPK1 promotes tau protein accumulation and its phosphorylation. Moreover, DAPK1 ablation in mice decreases tau protein stability and its phosphorylation at multiple AD-related sites. Originally identified as an important positive mediator of neuronal cell death, DAPK1 kinase activity-deficient mice are more efficient learners and have better spatial memory than wild type mice and DAPK1 is also genetically linked to late onset AD. These results suggest that aberrant DAPK1 activation might contribute to age-dependent neurodegeneration including AD. Our hypothesis guiding this study is that nanoprobes composed of DAPK1 inhibitors and targeting modulators (nanoinhibitors) can be an effective tool for accurate diagnosis of AD in its early state and their prognostic treatment. We propose to test DAPK1 inhibiting nanoprobes for biodistribution and pharmacokinetics using AD mouse models, as the lead compound in this proof of concept application. Thus, the overall goal in this supplemental application is to develop ultrasmall nanoinhibitors that are complexed with inhibitors targeted to both A? and tau, and evaluate the functionality and targetability in AD animal models.